SUMMARY Cytoplasmic nucleic acids alert the immune system to invading pathogens and trigger a robust type I interferon (IFN) response via activation of the STING (stimulator of interferon genes) receptor. The sensor for cytoplasmic nucleic acids was recently discovered to be cyclic GMP-AMP synthase, an enzyme that produces a unique cyclic dinucleotide second messenger, cGAMP, that serves as an agonist for the STING receptor. Aberrant activation of the cGAS-STING pathway is rapidly emerging as an important underlying cause of debilitating and sometimes fatal autoimmune disorders including Aicardi?Goutieres Syndrome (AGS), a monogenic encephalopathy that is usually fatal before adulthood, and systemic lupus erythematosus (SLE). Development of cGAS inhibitors is clearly a therapeutic strategy that should to be explored. There are no drugs approved specifically for any of the IFN-driven autoimmune diseases, and current IFN-targeted therapies in clinical development are mostly biologicals; e.g., antibodies against IFN? or the type IFN receptor. Inhibiting cGAS, the molecular trigger for type I IFN induction, is likely to be more efficient than blocking downstream targets, and a small molecule cGAS drug would have obvious advantages in terms of cost, dosing and CNS exposure. Aberrant activation of cGAS by dsDNA is the trigger for a constitutive type I IFN response, resulting in autoimmune diseases such AGS and SLE with no curative therapies. We developed an HTS-compatible cGAS enzymatic assay and used it to screen a 100k diversity library resulting in the identification of four novel cGAS inhibitor chemotypes. Following the primary screen, we performed iterative rounds of SAR informed by computational modeling and medicinal chemistry expertise to prioritize two chemotypes with different mechanisms of action, and to increase potency into the nanomolar range. During the course of this work we uncovered interplay between cGAS sensivity to activation by nucleic acids and its modulation by small molecules that could inform the development of drugs with a favorable therapeutic window. The goals of this Phase I proposal are to use structure-based design to improve the potency and ADME/PK properties of the lead chemotypes and to demonstrate target engagement and efficacy in cells. We have made substantial progress toward these goals, including generation of a high resolution co-crystal X-ray structure for one of the lead chemotypes and demonstration of specific inhibition of cGAS-driven IFN gene expression in human monocytes. In Phase I, we will continue to improve the potency and other drug like properties of the two lead chemotypes using structure-based design enabled by a more powerful computational method. In addition, we will use stem cell-derived neural cell models and primary human peripheral immune cells to test inhibitor efficacy in a disease-relevant context. Successful completion of these aims will clearly establish feasibility for Phase II in vivo efficacy studies in a mouse model for AGS, a key milestone for clinical translation. Blocking cGAS with a small molecule could lead to a curative therapy for AGS and would likely spur development of other drugs targeting the cGAS/STING pathway with potential impact on millions of people suffering from debilitating autoimmune diseases. This is a multidisciplinary lead discovery effort that will combine BellBrook's extensive enzymology and screening expertise, medicinal chemistry and translational research expertise from David Maloney and Matt Boxer of Nexus Discovery Advisors (Frederick, MD - LOS and bios attached), structural and biophysical expertise from XTAL BioStructures (Natick, MA, LOS attached) and computational chemistry expertise from SilcsBio (Baltimore, MD ? LOS attached). Dr. Keith Elkon, Co- director of the Center for Innate Immunity and Immune Disease and Head of Rheumatology at University of Washington School of Medicine is serving as a consultant (LOS and bio attached). Under a separate SBIR grant (R44GM123833) in collaboration with Dr. Elkon, BellBrook is developing methods for detecting cGAMP in cell and tissue samples to enable monitoring of cGAS inhibition in animal models, and eventually for stratification and monitoring of patients in clinical studies; e.g., AGS patients or SLE patients with high levels of cGAMP in PBMCs as candidates for cGAS inhibitors. The availability of a companion diagnostic would increase the potential medical impact and value of a cGAS inhibitor drug substantially.